Imidazolecarboxamides and their use as FAAH inhibitors

Abstract

A compound having a structure selected from the following: ##STR00001##
or a pharmaceutically acceptable salt thereof. The compound may be used as an inhibitor of fatty acid amide hydrolase.

Claims

1. A compound having a structure selected from the group consisting of: ##STR00022## or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of a hydrochloride salt, acetate salt, trifluoroacetate salt, methanesulfonate salt, 2-hydroxypropane-1,2,2-tricarboxylate salt, (2R,3R)-2,3-dihydroxysuccinate salt, phosphate salt, sulphate salt, benzoate salt, 2-hydroxy-benzoate salt, S-(+)-mandelate salt, S-()-malate salt, S-() pyroglutamate salt, pyruvate salt, p-toluenesulfonate salt, 1-R-()-camphorsulfonate salt, fumarate salt, maleate salt and oxalate salt.

3. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein the compound has the structure ##STR00023##

4. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein the compound has the structure ##STR00024##

5. The compound of claim 1 or the pharmaceutically acceptable salt thereof, wherein the compound has the structure ##STR00025##

6. A pharmaceutical composition comprising a compound according to claim 1, together with one or more pharmaceutically acceptable excipients.

7. The pharmaceutical composition of claim 6, further comprising one or more additional active pharmaceutical ingredients.

8. The pharmaceutical composition of claim 6, wherein the composition is formulated for oral administration.

9. The pharmaceutical composition of claim 6, wherein the composition is in the form of a sterile injectable preparation.

10. The pharmaceutical composition of claim 7, wherein the one or more additional active pharmaceutical ingredients are selected from the group consisting of anandamide, oleoyl ethanolamide, and palmitoyl ethanolamide.

11. A method of treatment of an eye condition selected from the group consisting of ocular hypertension and glaucoma, the method comprising the administration, to a subject in need of such treatment, of a therapeutically effective amount of a compound according to claim 1.

12. The method of claim 11, wherein the method comprises further administration of one or more additional active pharmaceutical ingredients, wherein the compound is administered simultaneously with or staggered with respect to, the one or more additional active pharmaceutical ingredients.

13. The method of claim 12, wherein the one or more additional active pharmaceutical ingredients are selected from the group consisting of anandamide, oleoyl ethanolamide and palmitoyl ethanolamide.

14. The method of claim 11, wherein the eye condition is ocular hypertension.

15. The method of claim 11, wherein the eye condition is glaucoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The invention will now be described in more detail by way of example only:

1. Synthetic Methodologies

(2) The methods used for synthesis of the compounds of the invention are illustrated by the general schemes and specific syntheses below. All compounds and intermediates were characterised by nuclear magnetic resonance (NMR). The starting materials and reagents used in preparing these compounds are available from commercial suppliers or can be prepared by methods obvious to those skilled in the art. These general schemes and specific syntheses are merely illustrative of methods by which the compounds of this invention can be synthesised, and various modifications to these schemes and syntheses can be made and will be suggested to one skilled in the art having referred to this disclosure.

(3) The compounds of the invention were characterised by melting point and NMR. NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer with solvent used as internal standard. 13C spectra were recorded at 150 MHz and 1H spectra were recorded at 600 MHz. Data are reported in the following order: approximate chemical shift (ppm), number of protons, multiplicity (br, broad; d, doublet; m, multiplet; s, singlet, t; triplet) and coupling constant (Hz).

(4) Room temperature in the following schemes means the temperature ranging from 20 C. to 25 C.

1.1. General Scheme for the Synthesis of N-Methyl-4-(3-(sulfamoylamino)phenyl)-N-(tetrahydro-2H-pyran-4-yl)-1H-imidazole-1-carboxamide (Compound 1)

(5) ##STR00003##

Phenyl 4-(3-nitrophenyl)-1H-imidazole-1-carboxylate

(6) ##STR00004##

(7) Phenyl carbonochloridate (3.2 mL, 25.4 mmol) was added to a stirred solution of 4-(3-nitrophenyl)-1H-imidazole (4 g, 21.1 mmol) and pyridine (2.0 mL, 25.4 mmol) in DCM (100 mL) at 0 C. The reaction mixture was allowed to stir at room temperature for 2 h. Water was added and the organic layer was separated, dried (MgSO.sub.4) and evaporated in vacuum to give a beige solid. The solid was then recrystallised from a mixture of propan-2-ol and DCM and the product was isolated as a beige solid. Phenyl 4-(3-nitrophenyl)-1H-imidazole-1-carboxylate (2.89 g, 44% yield).

N-Methyl-4-(3-nitrophenyl)-N-(tetrahydro-2H-pyran-4-yl)-1H-imidazole-1-carboxamide

(8) ##STR00005##

(9) A solution of N-methyltetrahydro-2H-pyran-4-amine (2.15 g, 18.7 mmol) in tetrahydrofuran (THF) (6 mL) was added to a stirred solution of phenyl 4-(3-nitrophenyl)-1H-imidazole-1-carboxylate (2.89 g, 9.3 mmol) in THF (40 mL) at room temperature. The yellow solution was allowed to stir at reflux overnight. The solvent was evaporated in vacuum and the product was recrystallised from propan-2-ol. N-methyl-4-(3-nitrophenyl)-N-(tetrahydro-2H-pyran-4-yl)-1H-imidazole-1-carboxamide (0.938 g, 30% yield).

4-(3-Aminophenyl)-N-methyl-N-(tetrahydro-2H-pyran-4-yl)-1H-imidazole-1-carboxamide

(10) ##STR00006##

(11) A mixture of ethanol (30.0 mL) and ethyl acetate (30 mL) was added to wet palladium (0.151 g, 0.142 mmol, 10% on activated charcoal) under an atmosphere of argon. To this mixture was added N-methyl-4-(3-nitrophenyl)-N-(tetrahydro-2H-pyran-4-yl)-1H-imidazole-1-carboxamide (0.938 g, 2.84 mmol) and the suspension was allowed to stir at room temperature overnight under an atmosphere of hydrogen. The resultant grey suspension was filtered through celite and the celite was washed with DCM. The filtrate was evaporated in vacuum and the colourless product was recrystallised from propan-2-ol.4-(3-Aminophenyl)-N-methyl-N-(tetrahydro-2H-pyran-4-yl)-1H-imidazole-1-carboxamide (0.695 g, 81% yield).

N-Methyl-4-(3-(sulfamoylamino)phenyl)-N-(tetrahydro-2H-pyran-4-yl)-1H-imidazole-1-carboxamide (Compound 1)

(12) ##STR00007##

(13) Sulfamoyl chloride (0.321 g, 2.78 mmol) was added to a stirred suspension of 4-(3-aminophenyl)-N-methyl-N-(tetrahydro-2H-pyran-4-yl)-1H-imidazole-1-carboxamide (0.695 g, 2.314 mmol) and triethylamine (0.481 mL, 3.47 mmol) in DCM (12 mL) at room temperature. The white suspension was allowed to stir at room temperature overnight. Water was added and the organic layer was diluted with a mixture of DCM/propan-2-ol 7:3. The organic layer was separated and the aqueous layer re-extracted. The combined organic layer was dried (MgSO.sub.4) and evaporated in vacuum to give a clear oil. The product was separated by column chromatography (silica, DCM/MeOH 2%, 5%, 10%) and was isolated as a colourless solid. The solid was triturated with propan-2-ol/DCM mixture. The solid was recrystallised twice from EtOH and the product was dried in high vacuum at 60 C. overnight. N-methyl-4-(3-(sulfamoylamino)phenyl)-N-(tetrahydro-2H-pyran-4-yl)-1H-imidazole-1-carboxamide (0.160 g, 18% yield). mp: 128 C.

(14) NMR (DMSO-d.sub.6):

(15) .sup.1H: 9.54 (1H, s), 8.14 (1H, s), 7.94 (1H, s), 7.64 (1H, s), 7.44 (1H, d, J=7.7 Hz), 7.27 (1H, t, J=7.6 Hz), 7.15 (2H, s), 7.05 (1H, d, J=8.2 Hz), 4.10 (1H, m), 3.93 (2H, dd, J=4.0, 11.3 Hz), 3.36 (2H, m), 2.95 (3H, s), 1.86 (2H, dq, J=4.1, 12.3 Hz), 1.70 (2H, d, J=12.0 Hz).

(16) .sup.13C: 151, 140.6, 139.9, 137.5, 134, 129, 118.6, 116.9, 114.6, 114.4, 66.3, 54.2, 31.6, 29.1.

1.2. General Scheme for the Synthesis of 4-(4-methoxy-3-methylphenyl)-N-methyl-N-(piperidin-4-yl)-1H-imidazole-1-carboxamide hydrochloride (Intermediate 1)

(17) ##STR00008##

1-(4-Methoxy-3-methylphenyl)ethanone

(18) ##STR00009##

(19) Dimethyl sulfate (17.50 mL, 183 mmol) was added to a stirred suspension of 1-(4-hydroxy-3-methylphenyl)ethanone (25 g, 166 mmol) and potassium carbonate (28.8 g, 208 mmol) in acetone (277 mL) at room temperature. The suspension was allowed to stir at reflux overnight. The solid was separated by filtration and was washed with acetone and the filtrate was evaporated in vacuum. The organic residue was dissolved in EtOAc and was washed with water. The organic layer was separated, dried (MgSO.sub.4) and evaporated in vacuum to give a yellow oil. Used without further purification. 1-(4-Methoxy-3-methylphenyl)ethanone (28.7 g).

2-Bromo-1-(4-methoxy-3-methylphenyl)ethanone

(20) ##STR00010##

(21) A solution of phenyltrimethylammonium tribromide (30.2 g, 80 mmol) in THF (122 mL) was added dropwise to a stirred solution of 1-(4-methoxy-3-methylphenyl)ethanone (12 g, 73.1 mmol) in THF (122 mL) at room temperature. The yellow suspension was allowed to stir at room temperature for 1 h. The solid was separated by filtration and was washed with THF. The filtrate was evaporated in vacuum and the organic residue was dissolved in EtOAc and was washed with water. The organic layer was dried (MgSO.sub.4) and evaporated in vacuum to give a violet oil. Used without further purification. 2-Bromo-1-(4-methoxy-3-methylphenyl)ethanone (27.9 g).

4-(4-methoxy-3-methylphenyl)-1H-imidazole

(22) ##STR00011##

(23) Water (4 mL) was added to a stirred suspension of 2-bromo-1-(4-methoxy-3-methylphenyl)ethanone (27.9 g, 115 mmol) and formamide (56.7 mL, 1423 mmol) at room temperature. The suspension was allowed to stir at 140 C. for 5 h. The mixture was poured into 200 mL of water to give a dark dense brown oil. The oil was separated by filtration and was washed with 1N HCl and the filtrate was basified with NaOH 50% to give a beige solid. The solid was separated by filtration and was washed with diethyl ether (5) to give an off-white solid. 4-(4-Methoxy-3-methylphenyl)-1H-imidazole (5.2 g, 24% yield).

tert-Butyl 4-(methylamino)piperidine-1-carboxylate

(24) ##STR00012##

(25) A solution of methanamine (38.0 mL, 442 mmol, 40% aqueous solution) in methanol (100 mL) was added to wet palladium (1.602 g, 1.506 mmol, 10% on activated charcoal) at room temperature under an atmosphere of argon. To this mixture was added tert-butyl 4-oxopiperidine-1-carboxylate (20 g, 100 mmol) portionwise and the mixture was allowed to stir at 50 C., 20 bar over 1 h. The suspension was flushed with argon and was filtered through celite and the celite was washed with DCM. The filtrate was evaporated in vacuum to give the product as a clear oil. The oil was dissolved in EtOAc and was washed with water. The organic layer was dried (MgSO.sub.4) and evaporated in vacuum to give a clear oil. Used without further purification. tert-Butyl 4-(methylamino)piperidine-1-carboxylate (20 g, 93% yield).

tert-Butyl 4-(chlorocarbonyl(methyl)amino)piperidine-1-carboxylate

(26) ##STR00013##

(27) A solution of tert-butyl 4-(methylamino)piperidine-1-carboxylate (20 g, 93 mmol) and Hunig's base (35.9 mL, 205 mmol) in THF (133 mL) was added dropwise to stirred phosgene (53.3 mL, 112 mmol, 20% solution in toluene) at 0 C. to give a white suspension. The mixture was allowed to stir at 0 C. for 10 min and at room temperature for 2 h. The suspension was poured into ice/water and the organic residue was extracted with EtOAc. The organic layer was separated and washed with 1N HCl solution. The organic layer was dried (MgSO.sub.4) and evaporated in vacuum to give a yellow oil. The oil was triturated with a mixture of PE and few drops of diethyl ether to give a colourless solid. The solid was separated by filtration and was washed with petroleum ether. tert-Butyl 4-(chlorocarbonyl(methyl)amino)piperidine-1-carboxylate (17.4 g, 67% yield).

tert-Butyl 4-(4-(4-methoxy-3-methylphenyl)-N-methyl-1H-imidazole-1-carboxamido)piperidine-1-carboxylate

(28) ##STR00014##

(29) Sodium hydride (1.313 g, 32.8 mmol, 60% dispersion in oil) was added portionwise to a stirred suspension of 4-(4-methoxy-3-methylphenyl)-1H-imidazole (5.15 g, 27.4 mmol) in THF (137 mL) at 0 C. The dark blue solution was allowed to stir at room temperature for 30 min and then tert-butyl 4-(chlorocarbonyl(methyl)amino)piperidine-1-carboxylate (11.36 g, 41.0 mmol) was added at 0 C. to give a dark solution. The mixture was allowed to stir at room temperature for 2 h. Water was added at 0 C. and the organic layer was diluted with a mixture of DCM/propan-2-ol 7:3. The organic layer was separated, dried (MgSO.sub.4) and evaporated in vacuum to give a beige solid. The solid was recrystallised from propan-2-ol tert-Butyl 4-(4-(4-methoxy-3-methylphenyl)-N-methyl-1H-imidazole-1-carboxamido)piperidine-1-carboxylate (9.39 g, 80% yield).

4-(4-Methoxy-3-methylphenyl)-N-methyl-N-(piperidin-4-yl)-1H-imidazole-1-carboxamide hydrochloride

(30) ##STR00015##

(31) TFA (30 mL) was carefully added to the residue tert-butyl 4-(4-(4-methoxy-3-methylphenyl)-N-methyl-1H-imidazole-1-carboxamido)piperidine-1-carboxylate (9.39 g, 21.91 mmol) at room temperature. The yellow solution was allowed to stir at room temperature for 1.5 h. The TFA was evaporated in vacuum and then was azeotroped twice with toluene. The yellow residue was then dissolved in ethyl acetate (30 mL) and a 2M solution of hydrogen chloride (32.9 mL, 65.7 mmol) in diethyl ether was added dropwise at 0 C. to give a white suspension. The mixture was allowed to stir at room temperature for 30 min and then the solid was separated by filtration and was washed with EtOAc. 4-(4-Methoxy-3-methylphenyl)-N-methyl-N-(piperidin-4-yl)-1H-imidazole-1-carboxamide hydrochloride (11.06 g).

1.3. General Scheme for the Synthesis of 4-(4-Hydroxy-3-methylphenyl)-N-methyl-N-(1-propionylpiperidin-4-yl)-1H-imidazole-1-carboxamide (Compound 2)

(32) ##STR00016##

4-(4-Methoxy-3-methylphenyl)-N-methyl-N-(1-propionylpiperidin-4-yl)-1H-imidazole-1-carboxamide

(33) ##STR00017##

(34) Propionyl chloride (0.287 mL, 3.29 mmol) was added to a stirred suspension of 4-(4-methoxy-3-methylphenyl)-N-methyl-N-(piperidin-4-yl)-1H-imidazole-1-carboxamide hydrochloride (Intermediate 1) (1 g, 2.74 mmol) and Hnig's base (0.957 mL, 5.48 mmol) in DCM (14 mL) at room temperature. The pink solution was allowed to stir at room temperature overnight. Water was added and the organic layer was diluted with DCM. The organic layer was separated, dried (MgSO.sub.4) and evaporated in vacuum to give an off-white solid. The solid was recrystallised from propan-2-ol. 4-(4-Methoxy-3-methylphenyl)-N-methyl-N-(1-propionylpiperidin-4-yl)-1H-imidazole-1-carboxamide (0.496 g, 45% yield).

4-(4-Hydroxy-3-methylphenyl)-N-methyl-N-(1-propionylpiperidin-4-yl)-1H-imidazole-1-carboxamide (Compound 2)

(35) ##STR00018##

(36) Boron tribromide (0.354 mL, 3.75 mmol) was added to a stirred suspension of 4-(4-methoxy-3-methylphenyl)-N-methyl-N-(1-propionylpiperidin-4-yl)-1H-imidazole-1-carboxamide (0.480 g, 1.248 mmol) in anhydrous DCM (4 mL) at 78 C. The suspension was allowed to stir at 78 C. for 15 min and at room temperature for 2 h. Water was added at 50 C. and then the organic layer was diluted with a mixture of DCM/propan-2-ol 7:3. The organic layer was separated; the aqueous layer was saturated with NaCl and re-extracted. The combined organic layer was dried (MgSO.sub.4) and evaporated in vacuum to give a clear oil. The product was separated by column chromatography (silica, DCM/MeOH 2%, 5%, 10%) and was isolated as a colourless solid. The solid was recrystallised from propan-2-ol. 4-(4-Hydroxy-3-methylphenyl)-N-methyl-N-(1-propionylpiperidin-4-yl)-1H-imidazole-1-carboxamide (0.22 g, 45% yield). mp: 232 C.

(37) NMR (DMSO-d.sub.6):

(38) .sup.1H: 9.34 (1H, s), 8.06 (1H, d, J=1.2 Hz), 7.77 (1H, d, J=1.2 Hz), 7.55 (1H, d, J=1.6 Hz), 7.47 (1H, dd, J=2, 8.3 Hz), 6.77 (1H, d, J=8.3 Hz), 4.53 (1H, d, J=12.5 Hz), 4.10 (1H, m), 3.95 (1H, d, J=13.5 Hz), 3.06 (1H, mt, J=13.0 Hz), 2.91 (3H, s), 2.56 (1H, mt, J=12.8 Hz), 2.34 (2H, q, J=7.5 Hz), 2.14 (3H, s), 1.76 (3H, m), 1.60 (1H, dq, J=4.3, 12.3 Hz), 0.98 (3H, t, J=7.5 Hz).

(39) .sup.13C: 171.1, 154.8, 151.1, 141.1, 137.3, 127.3, 124.2, 123.8, 123.4, 114.6, 112.3, 55.1, 43.9, 40.3, 31.6, 28.6, 28, 25.5, 16.1, 9.5.

1.4 General Scheme for the Synthesis of N-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-4-(4-hydroxy-3-methylphenyl)-N-methyl-1H-imidazole-1-carboxamide (Compound 3)

(40) ##STR00019##

N-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-4-(4-methoxy-3-methylphenyl)-N-methyl-1H-imidazole-1-carboxamide

(41) ##STR00020##

(42) Cyclopropanecarbonyl chloride (1.5 mL, 16.44 mmol) was added to a stirred suspension of 4-(4-methoxy-3-methylphenyl)-N-methyl-N-(piperidin-4-yl)-1H-imidazole-1-carboxamide hydrochloride (Intermediate 1) (5 g, 13.70 mmol) and Hunig's base (4.8 mL, 27.4 mmol) in DCM (70 mL) at room temperature. The pink solution was allowed to stir at room temperature overnight to give a pink suspension. Water was added and the organic layer was diluted with DCM. The organic layer was separated, dried (MgSO.sub.4) and evaporated in vacuum to give clear oil that solidified into an off-white solid. The solid was recrystallised from propan-2-ol. N-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-4-(4-methoxy-3-methylphenyl)-N-methyl-1H-imidazole-1-carboxamide (2.96 g, 55% yield).

N-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-4-(4-hydroxy-3-methylphenyl)-N-methyl-1H-imidazole-1-carboxamide (Compound 3)

(43) ##STR00021##

(44) Boron tribromide (0.358 mL, 3.78 mmol) was added to a stirred suspension of N-(1-(cyclopropanecarbonyl)piperidin-4-yl)-4-(4-methoxy-3-methylphenyl)-N-methyl-1H-imidazole-1-carboxamide (0.500 g, 1.261 mmol) in anhydrous DCM (4.20 mL) at 78 C. The suspension was allowed to stir at 78 C. for 15 min and at room temperature for 2 h. Water was added at 50 C. and then the organic layer was diluted with a mixture of DCM/propan-2-ol 7:3. The organic layer was separated; the aqueous layer was saturated with NaCl and re-extracted. The combined organic layer was dried (MgSO.sub.4) and evaporated in vacuum to give a clear oil. The product was separated by column chromatography (silica, DCM/MeOH 2%, 5%, 10%) and was isolated as a colourless solid. The solid was recrystallised from a mixture of propan-2-ol and DCM. N-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-4-(4-hydroxy-3-methylphenyl)-N-methyl-1H-imidazole-1-carboxamide (0.289 g, 57% yield). mp: 204 C.

(45) NMR (DMSO-d.sub.6):

(46) .sup.1H: 9.32 (1H, s), 8.06 (1H, d, J=1.2 Hz), 7.76 (1H, d, J=1.2 Hz), 7.55 (1H, d, J=1.6 Hz), 7.47 (1H, dd, J=2, 8.2 Hz), 6.77 (1H, d, J=8.2 Hz), 4.51 (1H, d, J=12.0 Hz), 4.37 (1H, d, J=13.0 Hz), 4.13 (1H, m), 3.15 (1H, t, J=13.0 Hz), 2.92 (3H, s), 2.61 (1H, mt, J=13.0 Hz), 2.14 (3H, s), 2.0 (1H, m), 1.85 (1H, md), 1.77 (2H, m), 1.63 (1H, mq), 0.8-0.66 (4H, m).

(47) .sup.13C: 170.8, 154.8, 151.2, 141.1, 137.3, 127.3, 124.2, 123.8, 123.4, 114.6, 112.4, 55.2, 44, 40.8, 31.7, 28.9, 28, 16.1, 10.3, 7, 6.9.

2. Biological Efficacy

(48) All animal procedures were conducted in strict adherence to the European Directive for Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (86/609CEE) and Portuguese legislation (Decreto-Lei 129/92, Portarias 1005/92 e 1131/97). The number of animals used was the minimum possible in compliance with current regulations and scientific integrity.

(49) In vivo testing was performed according to the protocol described below. BRh (brain homogenate) indicates inhibition in central nervous tissue, in this case, brain, and LVh (liver homogenate) indicates inhibition in peripheral tissue, in this case, liver. The controls were the reaction mix minus the test compounds. Therefore, a low value for the test compound indicates a strong inhibitor. A value of 100 indicates that no measurable inhibition took place.

(50) In Vivo Protocols

(51) Experiments in Mice

(52) Animal Treatment

(53) The animals used for experiments were male NMRI mice (weighing 27-44 g) obtained from Interfauna Ibrica (Spain). Mice were kept 5 per cage, under controlled environmental conditions (12 hr light/dark cycle and room temperature 221 C.). Food and tap water were allowed ad libitum and the experiments were all carried out during daylight hours.

(54) Animals were always fasted overnight before administration of compounds.

(55) Animals were administered the appropriate dose of the compound of the invention via oral route (8 ml/kg; compound suspended in 0.5% carboxymethylcellulose (CMC) or solubilized in water) or vehicle (controls) using animal feeding stainless steel curve needles (Perfectum, U.S.A.). Fifteen minutes before sacrifice animal were anesthetized with pentobarbital 60 mg/kg administered intraperitoneally. A fragment of liver and brain without cerebellum were removed and put in plastic vials containing membrane buffer (3 mM MgCl.sub.2, 1 mM EDTA, 50 mM Tris HCl pH 7.4). Tissues were stored at 30 C. until analysis.

(56) Reagents and Solutions

(57) Anandamide [ethanolamine-1-.sup.3H-] (40-60 Ci/mmol) was obtained from American Radiochemicals. All other reagents were obtained from Sigma-Aldrich. Optiphase Supermix was obtained from Perkin Elmer and activated charcoal was obtained from Sigma-Aldrich.

(58) Tissue Preparation

(59) Tissues were thawed on ice and were homogenized in 10 volumes of membrane buffer (3 mM MgCl.sub.2, 1 mM EDTA, 50 mM Tris HCl pH 7.4) with either Potter-Elvejhem (brains8 strokes at 500 rpm) or Heidolph Diax (livers2 strokes at position 5 for 20 sec with 30 sec pauses).

(60) Total protein in tissues was determined with the BioRad Protein Assay (BioRad) using a standard curve of BSA (50-250 g/ml).

(61) Enzymatic Assay

(62) Reaction mix (total volume of 200 l) contained: 2 M AEA (2 M AEA+5 nM .sup.3H-AEA), 0.1% fatty acid free BSA, 15 g (brain) or 5 g (liver) protein, in 1 mM EDTA, 10 mM Tris pH 7.6. After a 15 min pre-incubation period at 37 C., reaction was started by the addition of the substrate solution (cold AEA+radiolabelled AEA+BSA). Reaction was carried out for 10 min (brain) or 7 min (liver) before termination by the addition of 400 l activated charcoal suspension (8 g charcoal in 32 ml 0.5 M HCl in continuous agitation). After a 30 min incubation period at room temperature with agitation, charcoal was sedimented by centrifugation in microfuge (10 min at 13000 rpm). 200 l of the supernatant were added to 800 l Optiphase Supermix scintillation cocktail previously distributed in 24-well plates. Counts per minute (cpm) were determined in a MicrobetaTriLux scintillation counter.

(63) In each assay blanks (without protein) were prepared.

(64) The percentage of remaining enzymatic activity was calculated with respect to controls and after blank subtraction.

(65) Experiments in Rats

(66) Animal Treatment

(67) Male Wistar rats (body weight range: 190-230 g) were obtained from Harlan (Spain). Rats were kept 5 per cage, under controlled environmental conditions (12 hr light/dark cycle and room temperature 221 C.). Food and tap water were allowed ad libitum and the experiments were all carried out during daylight hours.

(68) Rats were administered the appropriate dose of compound of the invention via gavage (administration volume=4 ml/kg body weight) using animal feeding stainless steel curve needles (Perfectum, U.S.A.). Vehicle was 0.5% CMC in Milli Q water. Rats were fasted at least 15 h before experiments.

(69) Fifteen minutes before sacrifice animals were anesthetized with pentobarbital i.p. 60 mg/kg body weight. Liver biopsies and brain samples (without cerebellum) were collected and placed in a plastic vial containing membrane buffer (3 mM MgCl2, 1 mM EDTA, 50 mM Tris HCl pH 7.4) and, in the case of liver samples, glass beads (2.5 mm BioSpec Products). Tissues were stored at 20 C. until analysis.

(70) Reagents and Solutions

(71) Anandamide [ethanolamine-1-3H-] was obtained from American Radiochemicals (specific activity of 60 Ci/mmol). All other reagents were obtained from Sigma-Aldrich. Optiphase Supermix was obtained from Perkin Elmer.

(72) Tissue Preparation

(73) Tissues were thawed on ice; livers were homogenized in a Precellys 24 Dual Tissue Homogenizer (Bertin Technologies) for 2 cycles of 5 sec with an interval of 5 min in ice and brains were homogenized with Heidolph Silent Crusher M (probe 8 F/M) for about 45 sec at maximum velocity. Total protein in homogenates was determined with the BioRad Protein Assay (BioRad) using a standard curve of BSA (50-250 g/ml).

(74) Enzymatic Assay

(75) Reaction mix (total volume of 200 l) contained: 2 M AEA (2 M AEA+5 nM 3H-AEA), 0.1% fatty acid free BSA, 15 g (brain) or 1.5 g (liver) protein, in 1 mM EDTA, 10 mM Tris pH 7.6. After 15 minutes pre-incubation at 37 C. reaction was started by the addition of the substrate solution (cold AEA+radiolabelled AEA+BSA). Reaction was carried out for 7 minutes for liver samples and for 10 min for brain samples and terminated by addition of 400 L chloroform:methanol (1:1, v/v) solution. Reaction samples were vortex twice, left on ice for 5 minutes and then centrifuged in microfuge (7 minutes, 7000 rpm). Two-hundred l of supernatants were added to 800 l Optiphase Supermix scintillation cocktail previously distributed in 24-well plates. Counts per minute (cpm) were determined in a Microbeta TriLux scintillation counter. In each assay blank samples (without protein) were prepared. The percentage of remaining enzymatic activity was calculated in respect to controls and after blank subtraction.

(76) CYPs Metabolic Stability Assay

(77) Stability of the test compounds was performed in MLM (mouse liver microsomes) or HLM (human liver microsomes) in the presence and in the absence of NADPH.

(78) The stability was measured using the incubation mixture (100 l total volume) contained 1 mg/ml total protein, MgCl.sub.2 5 mM and 50 mM K-phosphate buffer. Samples were incubated in the presence and in the absence of NADPH 1 mM. Reactions were pre-incubated 5 min and the reaction initiated with the compound under test (5 M for HLM and 50 M for MLM). Samples were incubated for 60 min in a shaking water bath at 37 C. The reaction was stopped by adding 100 l of acetonitrile. Samples were then centrifuged, filtered and supernatant injected in HLPC-MSD. Test compounds were dissolved in DMSO and the final concentration of DMSO in the reaction was below 0.5% (v/v). At T0 acetonitrile was added before adding the compound. All experiments were performed with samples in duplicate.

(79) Compounds Tested:

(80) Compound 1=(N-methyl-4-(3-(sulfamoylamino)phenyl)-N-(tetrahydro-2H-pyran-4-yl)-1H-imidazole-1-carboxamide). Compound 2=((4-(4-Hydroxy-3-methylphenyl)-N-methyl-N-(1-propionylpiperidin-4-yl)-1H-imidazole-1-carboxamide). Compound 3=((N-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-4-(4-hydroxy-3-methylphenyl)-N-methyl-1H-imidazole-1-carboxamide).

(81) TABLE-US-00001 FAAH Activity (%) FAAH Activity (%) Br .Math. h .Math. 3 mg/kg .Math. 8 h .Math. po Lv .Math. h .Math. 3 mg/kg .Math. 8 h .Math. po Compound 1 117 4.9 Compound 2 81.7 1.5 Compound 3 91.7 1.8

(82) As can be seen from the above table, compounds 1, 2 and 3 are all relatively potent compounds in terms of FAAH inhibition in the liver.

(83) Peripheral selectivity can be calculated by dividing the FAAH activity in the liver by the FAAH activity in the brain. When doing this, a lower number shows a compound is peripherally more selective. The results are given in the table below:

(84) TABLE-US-00002 Peripheral Selectivity Compound 1 0.042 Compound 2 0.018 Compound 3 0.020

(85) These results show that compounds 2 and 3 are the most peripherally selective compounds but that all the compounds show relatively high peripheral selectivity.

(86) Additional data relating to the activity of FAAH at various concentrations for the compounds are given in the table below:

(87) TABLE-US-00003 FAAH Activity (%) mouse Liver 1 h 8 h 1 0.1 0.3 0.1 0.03 0.01 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Compound 1 8.5 36.1 Compound 2 2.3 21.9 5.6 6.7 42.9 48.2 Compound 3 37.9 7.2 10.7 53.0 56.3

(88) As can be seen above, compounds 2 and 3 are the most potent as they inhibit FAAH activity even at a relatively low dose. However, all the compounds are relatively potent.

(89) Further, similar experiments were conducted in rats which gave the following results:

(90) TABLE-US-00004 FAAH Activity (%) rat Liver Dose: 0.1 mg/kg Dose: 1 mg/k9 1 h 8 h 1 h 8 h 24 h Compound 1 9.8 4.8 22.7 Compound 2 78.2 59.1 25.7 18.2 17.8 Compound 3 83.5 62.5

(91) As can be seen above, all the compounds show relatively good inhibition in rat liver and are relatively potent.

(92) Metabolic Stability

(93) The below table shows the metabolic stability of the compounds. The stability data are given as % of remaining compound after 1 h exposure to MLM or HLM. 100% means no metabolic reaction at all and 0% corresponds to full enzymatic degradation. CYP refers to the absence of cofactor (NADPH) which is essential for CYP metabolic reactions. Therefore CYP can be regarded as control value. CYP+ refers to the presence of cofactor and the enzymatic degradation may take place according to the stability of the test compound. As can be seen, all the compounds are metabolically stable.

(94) TABLE-US-00005 Metabolic Stability (% of Remaining) Mouse Human CYP+ CYP CYP+ CYP Compound 1 93 100 96 102 Compound 2 86 83 100 94 Compound 3 98 91 99 102